Rifamycin analogs and uses thereof

ABSTRACT

The present invention features rifamycin analogs and methods of using these compounds to treat a variety of microbial infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/750,902 filed on Dec. 14, 2005, herein incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the field of antimicrobial agents.

The use of antibiotics by humans can be seen as an evolutionaryexperiment of enormous magnitude, a window from which to viewnot-quite-natural selection operating in real time. Within 50 years, thenumber of species and strains of pathogenic and commensal bacteriaresistant to antibiotics and the number of antibiotics to which they areresistant has increased virtually monotonically world-wide. As a result,infections that had been readily treatable by chemotherapy may no longerbe so. It is clear that the evolution and spread of resistance can beattributed to the use and overuse of antibiotics. Increased resistanceof bacterial infections to antibiotic treatment has been extensivelydocumented and has now become a generally recognized medical problem,particularly with nosocomial infections. See, for example, Jones et al.,Diagn. Microbiol. Infect. Dis. 31:379-388, 1998; Murray, Adv. Intern.Med. 42:339-367, 1997; and Nakae, Microbiologia 13:273-284, 1997.

Throughout the developed world there is public and governmental concernabout the increasing prevalence of antimicrobial resistance tochemotherapy in bacteria that cause diseases in humans. Many pathogensexist for which there are few effective treatments, and the number ofstrains resistant to available drugs is continually increasing. Newantimicrobial agents and improved methods are thus needed for thetreatment and prevention of infections by such pathogens.

SUMMARY OF THE INVENTION

The present invention features rifamycin analogs that can be used astherapeutics for treating a variety of microbial infections.

Accordingly in a first aspect, the invention features a compound havingthe formula:

In formula I, A is H, OH, O—(C₁₋₆ alkyl), O—(C₁₋₄ alkaryl), O—(C₆₋₁₂aryl), O— (C₁₋₉ heteroaryl), or O—(C₁₋₄ alkheteroaryl); W is O, S, orNR¹, wherein R¹ is H, C₁₋₆ alkyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl; Xis H or COR², wherein R² is C₁₋₆ alkyl, which can be substituted with1-5 OH groups, O—(C₃₋₇ alkyl), which can be substituted with 1-4 OHgroups, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄alkheteroaryl, wherein each alkyl carbon is bonded to to no more thanone oxygen atom; Y is H, Hal, or OR^(Y3), wherein R^(Y3) is C₁₋₆ alkyl,C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl; Z isH, Hal, or OR^(Z3), wherein R^(Z3) is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl; and

R⁴ has the formula:

where

R⁵ is H, C₁₋₆ alkyl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, COR¹⁰, CO₂R¹¹,CONR¹⁰R¹¹ CSR¹⁰, COSR¹¹, CSOR¹¹, CSNR¹⁰R¹¹, SO₂R¹¹, or SO₂NR¹⁰R¹¹,wherein R¹⁰ is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl,or C₁₋₄ alkheteroaryl, R¹¹ is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉heteroaryl, or C₁₋₄ alkheteroaryl, or R¹⁰ and R¹¹ together form a C₂₋₆linkage, optionally containing a non-vicinal O;

R⁶ is H, C₁₋₆ alkyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl;

R⁷is H, C₁ ₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₉ heteroaryl, C₂₋₉ heterocyclyl,C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, OR¹², or NR¹²R¹³, where R¹² is H, C₁₋₆alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl,R¹³ is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄alkheteroaryl, or R¹² and R¹³ together form a C₂₋₆ linkage, optionallycontaining a non-vicinal O;

T is O, S, NR⁵, or a bond;

each of R⁸ and R⁹ is, independently, H, C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₉heteroaryl, C₂₋₉ heterocyclyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, orR⁸ and R⁵ together form a 3-8-membered ring, with the ring optionallycontaining a non-vicinal oxygen;

and each of r and s is, independently, 1 or 2.

In one embodiment, the compound of formula I is one of the followingcompounds:

The invention also features pharmaceutical compositions that include acompound of formula I and a pharmaceutically acceptable carrier ordiluent.

In another aspect, the invention features a method of killing, treating,or preventing a microbial infection in an animal, preferably a mammal,and most preferably a human, that includes administering to the animal acompound or pharmaceutical composition of the invention. The inventionfurther features treating or preventing diseases associated with suchmicrobial infections. Such methods of treatment or prevention mayinclude the oral, topical, intravenous, intramuscular, or subcutaneneousadministration of a composition of the invention.

The invention also features a method for treating or preventing thedevelopment of an atherosclerosis-associated disease in a patient byadministering to the patient a compound of formula I in an amounteffective to treat or prevent the development of theatherosclerosis-associated disease in the patient. The patient istypically diagnosed as having the atherosclerosis-associated disease (orbeing at increased risk of developing the disease) or as havingmacrophages or foam cells infected with C. pneumoniae prior to theadministration of a compound of formula I.

The invention also features a method of reducing the level of C-reactiveprotein in a patient in need thereof by administering to the patient acompound of formula I in an amount effective to reduce the level ofC-reactive protein in the patient. In one embodiment, the patient hasnot been diagnosed as having a bacterial infection. In anotherembodiment, the patient has been diagnosed as having macrophages or foamcells infected with C. pneumoniae.

The invention also features a method for reducing C. pneumoniaereplication in macrophages or foam cells in a patient in need thereof byadministering a compound of formula I to the patient in an amounteffective to reduce C. pneumoniae replication in macrophages or foamcells in the patient.

The invention also features a method for treating a persistent C.pneumoniae infection in macrophages or foam cells in a patient byadministering a compound of formula I to the patient in an amounteffective to treat the C. pneumoniae infection in macrophages or foamcells in the patient.

The invention also features a method for treating a chronic diseaseassociated with an infection of C. pneumoniae by administering acompound of formula I to the patient in an amount effective to treat theinfection.

In any of the foregoing aspects, the dosage of a compound of formula Iis normally about 0.001 to 1000 mg/day. The compound may be given daily(e.g., a single oral dose of 2.5 to 25 mg/day) or less frequently (e.g.,a single oral dose of 5, 12.5, or 25 mg/week). Treatment may be for oneday to one year, or longer. In one embodiment, a compound of formula Iis administered at an initial dose of 2.5 to 100 mg for one to sevenconsecutive days, followed by a maintenance dose of 0.005 to 10 mg onceevery one to seven days for one month, one year, or even for the life ofthe patient.

If desired, a compound of formula I may be administered in conjunctionwith one or more additional agents such as anti-inflammatory agents(e.g., non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen,diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam,nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin,celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodiumand magnesium salicylate) and steroids (e.g., cortisone, dexamethasone,hydrocortisone, methylprednisolone, prednisolone, prednisone,triamcinolone)), antibacterial agents (e.g., azithromycin,clarithromycin, erythromycin, gatifloxacin, levofloxacin, amoxicillin,metronidazole, penicillin G, penicillin V, methicillin, oxacillin,cloxacillin, dicloxacillin, nafcillin, ampicillin, carbenicillin,ticarcillin, mezlocillin, piperacillin, azlocillin, temocillin,cepalothin, cephapirin, cephradine, cephaloridine, cefazolin,cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef,cefoxitin, cefmatozole, cefotaxime, ceftizoxime, ceftriaxone,cefoperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir,cefpirome, cefepime, BAL5788, BAL9141, imipenem, ertapenem, meropenem,astreonam, clavulanate, sulbactam, tazobactam, streptomycin, neomycin,kanamycin, paromycin, gentamicin, tobramycin, amikacin, netilmicin,spectinomycin, sisomicin, dibekalin, isepamicin, tetracycline,chlortetracycline, demeclocycline, minocycline, oxytetracycline,methacycline, doxycycline, telithromycin, ABT-773, lincomycin,clindamycin, vancomycin, oritavancin, dalbavancin, teicoplanin,quinupristin and dalfopristin, sulphanilamide, para-aminobenzoic acid,sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfathalidine,linezolid, nalidixic acid, oxolinic acid, norfloxacin, perfloxacin,enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin,fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin,moxifloxacin, gemifloxacin, sitafloxacin, daptomycin, garenoxacin,ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, ortrimethoprim), platelet aggregation inhibitors (e.g., abciximab,aspirin, cilostazol, clopidogrel, dipyridamole, eptifibatide,ticlopidine, or tirofiban), anticoagulants (e.g., dalteparin,danaparoid, enoxaparin, heparin, tinzaparin, or warfarin), antipyretics(e.g., acetaminophen), or lipid lowering agents (e.g., cholestyramine,colestipol, nicotinic acid, gemfibrozil, probucol, ezetimibe, or statinssuch as atorvastatin, rosuvastatin, lovastatin simvastatin, pravastatin,cerivastatin, and fluvastatin). These additional agents may beadministered within 14 days, 7 days, 1 day, 12 hours, or 1 hour ofadministration of a compound of formula I, or simultaneously therewith.The additional therapeutic agents may be present in the same ordifferent pharmaceutical compositions as the compound of formula I. Whenpresent in different pharmaceutical compositions, different routes ofadministration may be used. For example, a compound of formula I may beadministered orally, while a second agent may be administered byintravenous, intramuscular, or subcutaneous injection.

The invention also features a stent coated with a compound of formula I.

The stent can be, e.g., a wire mesh tube used to hold open an artery.Stents are typically inserted following angioplasty.

The invention also features methods and compositions for treating orpreventing an ear infection in a patient by orally administering ortopically administering to the affected otic area (e.g., the tympanicmembrane or the external auditory canal of the ear) of the patient apharmaceutical composition including a therapeutically effective amountof a compound of formula I. The compositions and methods of theinvention can also be used to treat or prevent infections that resultfrom surgery.

The invention also features a pharmaceutical composition suitable fortopical administration to the ear of a patient containing a compound offormula I and a pharmaceutically-acceptable excipient, administered at adose capable of reducing the infection in the patient. According to thisinvention, the compound of formula I can be in the amount between 0.001%and 5% weight/volume (w/v), preferably 0.01% and 3% w/v, more preferably0.1% and 1% w/v, or most preferably 0.1% and 0.4% w/v. The compound offormula I can also be impregnated in a porous media (for example, an earwick such as a sponge, gauze, cotton, or hydrocellulose), which issuitable for insertion into the ear of a patient. If desired, thecomposition may also include one or more penetration enhancers (e.g.,alcohols, polyols, sulfoxides, esters, ketones, amides, oleates,surfactants, alkanoic acids, lactam compounds, alkanols, or admixturesthereof).

In another aspect, the invention also features a method for treating orpreventing the development of an ear infection in a patient using acomposition described above. A compound of formula I can be administeredto the infected ear by means of drops or by the insertion of acompound-impregnated porous media into the external ear canal to thetympanic membrane. Ear infections that can be treated using the methodsand composition of the invention include otitis media and otitis extema.Types of otitis media amenable to treatment include, for example, acuteotitis media, otitis media with effusion, and chronic otitis media.Types of otitis extema include acute otitis externa, chronic otitisextema, and malignant otitis externa.

A compound of the invention can also be administered to the ear (e.g.,the tympanic membrane or the external auditory canal of the ear) totreat or prevent bacterial infections associated with otitis media(e.g., an infection of H. influenza, M. catarhalis, or S. pneumoniae) orotitis externa (e.g., an infection of S. intermedius, Streptococcus spp.Pseudomonas spp., Proteus spp., or E. coli).

The methods and compositions of the invention are also useful to treatinfections associated with otic surgical procedures such astympanoplasty, stapedectomy, removal of tumors, or cochlear implantsurgery. The compositions may also be used prophylactically, prior totherapies or conditions that can cause ear infections. Compositionscontaining a compound of formula I can therefore be applied to an areaof the ear to which the surgical intervention will be performed, withinat least seven days (before or after) of the surgical intervention. Whentreating a patient affected with otitis externa, an acidificationtherapy involving the administration of an acetic acid solution to theear of the patient may also be performed.

Typically, patients are administered one to four drops of a compound ofthe invention in a total amount between 0.001% and 5% w/v, preferably0.01% and 3% w/v, more preferably 0.1% and 1% w/v, or most preferably0.1% and 0.4% w/v. The composition may be given daily (e.g., once,twice, three times, or four times daily) or less frequently (e.g., onceevery other day, or once or twice weekly). Treatment may be for 1 to 21days, desirably 1 to 14 days, or even 3 to 7 days. Additionaltherapeutic agents, such as anti-inflammatory agents (e.g.,non-steroidal anti-inflammatory or steroid), anesthetics, zinc salts, orother antimicrobial agents, can also be administered with a compound ofthe invention. Non-steroidal anti-inflammatory agents include, forexample, detoprofen, diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, indomethacin, ketoprofen, mechlofenameate, mefenamic acid,meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac,tolmeting, celecoxib, rofecoxib, choline salicylate, salsate, sodiumsalicylate, magnesium salicylate, aspirin, ibuprofen, paracetamol,acetaminophen, and pseudoephedrine and steroids include, for example,hydrocortisone, prednisone, fluprednisolone, triamcinolone,dexamethasone, betamethasone, cortisone, prednilosone,methylprednisolone, fluocinolone acetonide, flurandrenolone acetonide,and fluorometholone. Anesthetics according to the invention can be, forexample, benzocaine, butamben picrate, tetracaine, dibucaine,prilocaine, etidocaine, mepivacaine, bupivicaine, and lidocaine. A zincsalt can be zinc sulfate, zinc chloride, zinc acetate, zinc phenolsulfonate, zinc borate, zinc bromide, zinc nitrate, zincglycerophosphate, zinc benzoate, zinc carbonate, zinc citrate, zinchexafluorosilicate, zinc diacetate trihydrate, zinc oxide, zincperoxide, zinc salicylate, zinc silicate, zinc stannate, zinc tannate,zinc titanate, zinc tetrafluoroborate, zinc gluconate, and zincglycinate, and antimicrobial agents according to the invention include,for example, azithromycin, clarithromycin, erythromycin, gatifloxacin,levofloxacin, amoxicillin, metronidazole, penicillin G, penicillin V,methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin,ampicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin,azlocillin, temocillin, cepalothin, cephapirin, cephradine,cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin,cefprozil, cefaclor, loracarbef, cefoxitin, cefmatozole, cefotaxime,ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime,cefpodoxime, ceftibuten, cefdinir, cefpirome, cefepime, BAL5788,BAL9141, imipenem, ertapenem, meropenem, astreonam, clavulanate,sulbactam, tazobactam, streptomycin, neomycin, kanamycin, paromycin,gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin,dibekalin, isepamicin, tetracycline, chlortetracycline, demeclocycline,minocycline, oxytetracycline, methacycline, doxycycline, telithromycin,ABT-773, lincomycin, clindamycin, vancomycin, oritavancin, dalbavancin,teicoplanin, quinupristin and dalfopristin, sulphanilamide,para-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole,sulfathalidine, linezolid, nalidixic acid, oxolinic acid, norfloxacin,perfloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin,lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin,clinafloxacin, moxifloxacin, gemifloxacin, sitafloxacin, daptomycin,garenoxacin, ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, ortrimethoprim. These additional therapeutic agents can be present in thesame or different pharmaceutical compositions as a compound of formulaI. When a therapeutic agent is present in a different pharmaceuticalcomposition, different routes of administration may be used. A compoundof formula I and the second therapeutic agent, for example, may also beadministered within 24 hours of each other, and an anti-inflammatoryagent, for example, may be administered orally, or by intravenous,intramuscular, or subcutaneous injection.

To increase the efficacy of a topically administered compositioncontaining a compound of the invention, it is desirable that the amountof debris and granulation tissue are reduced at least once a day in theinfected ear of the patient and at least one hour prior to theadministration of a compound of the invention. Debris can be removed,for example, by suction, irrigation with a solution containing hydrogenperoxide, cauterization, or by manual techniques employingmicroinstruments and microscope. Reduction in the amount of granulationtissue in the infected ear may be performed by means of cauterizing, orby the administration of a steroid.

The invention also features a pharmaceutical pack containing (i) acompound of formula I in an amount effective to treat a patient havingan ear infection; and (ii) instructions for administering the compoundto the ear of a patient. The invention also features a compositioncontaining a compound of formula I and a pharmaceutical excipientsuitable for topical administration to the ear. If desired, anapplicator for applying the composition to the ear may also be included.Desirably, the composition contains a compound of formula I in theamount between 0.001% and 5% weight/volume (w/v), preferably 0.01% and3% w/v, more preferably 0.1% and 1% w/v, or most preferably 0.1% and0.4% w/v and is present in amounts sufficient to treat for at least 1,3, 5, 7, 10, 14, or 21 days. A penetration enhancer may also be added(e.g., alcohols, polyols, sulfoxides, esters, ketones, amides, oleates,surfactants, alkanoic acids, lactam compounds, alkanols, or admixturesthereof).

The invention also features a method for treating chronic gastritis,gastric ulcer, or duodenal ulcer associated with an infection of H.pylori, or preventing the disease or infection, in a patient. The methodincludes the step of administering, for example, orally to the patient,an effective amount of a compound of formula I to treat the patient. Thecompound is normally administered at about 0.1 to 1000 mg/day (desirablyabout 1 to 100 mg/day, more desirably about 1 to 50 mg/day, and evenmore desirably about 1 to 25 mg/day). The compound may be given daily(e.g., once, twice, three times, or four times daily) or less frequently(e.g., once every other day, or once or twice weekly). Treatment may befor 1 to 21 days, desirably 1 to 14 days or even 3 to 7 days. Ifdesirable, a compound of the invention can be administered with a protonpump inhibitor (e.g., omeprazole, esomeprazole, lansoprazole,leminoprazole, pantoprazole, or robeprazole), and/or bismuth preparation(e.g., colloidal bismuth subcitrate or bismuth subsalicylate).

The invention also features a pharmaceutical pack including (i) acompound of formula I in an amount effective to treat chronic gastritis,gastric ulcer, or duodenal ulcer associated with an infection of H.pylori in a patient; and (ii) instructions for administering thecompound to the patient. Desirably, the compound is in unit amounts ofbetween 0.1 and 1000 mg (e.g., between 1 and 50 mg or between 5 and 50mg), and is present in amounts sufficient to treat for at least 1, 3, 5,7, 10, 14, or 21 days. The pack may optionally include a proton pumpinhibitor and/or bismuth preparation. In one embodiment, a compound offormula I is in a pharmaceutical composition with the proton pumpinhibitor and/or bismuth preparation.

The invention also features a method for treating a patient havingantibiotic-associated bacterial diarrhea or an infection of C.difficile, or preventing the disease or infection in the patient. Themethod includes the step of orally administering to the patient aneffective amount of a compound of formula I to treat the patient. Thecompound is normally administered at about 0.1 to 1000 mg/day (desirablyabout 1 to 100 mg/day, more desirably about 1 to 50 mg/day, and evenmore desirably about 1 to 25 mg/day). The compound may be given daily(e.g., once, twice, three times, or four times daily) or less frequently(e.g., once every other day, or once or twice weekly). Treatment may befor 1 to 21 days, desirably 1 to 14 days or even 3 to 7 days. In oneembodiment, a compound of the invention is administered at an initialdose of between 5 and 100 mg, followed by subsequent doses of between 1and 50 mg for 3 to 7 days. A single dose (e.g., in a dosage of between 5and 50 mg) can also be employed in the method of the invention. Ifdesirable, a compound of formula I can be administered with a secondantibiotic (e.g., metronidazole or vancomycin), either simultaneously orsequentially.

The invention also features a pharmaceutical pack including (i) acompound of formula I in an amount effective to treat a patient havingantibiotic-associated bacterial diarrhea or an infection of C.difficile; and (ii) instructions for administering the compound to thepatient for treating or preventing a C. difficile infection. Desirably,the compound is in unit amounts of between 1 and 1000 mg (e.g., between1 and 50 mg or between 5 and 50 mg), and is present in amountssufficient to treat for at least 1, 3, 5, 7, 10, 14, or 21 days.

The invention features a method for treating a patient having aninfection of Chlamydia trachomatis. The method includes the step ofadministering to the patient a compound of formula I in an amounteffective to treat the patient. In one embodiment, the patient isadministered the compound as a single oral dose.

The invention also features a pharmaceutical pack that includes (i) asingle oral dose of a compound of formula I in an amount effective totreat a patient having an infection of C. trachomatis or N. gonorrhoeae;and (ii) instructions for administering the single oral dose to thepatient. Desirably, the dose is in an amount between 0.1 and 100 mg(e.g., between 1 and 50 mg or between 5 and 25 mg).

The invention also features a method of treating a patient having achronic disease associated with a bacterial infection caused by bacteriacapable of establishing a cryptic phase. This method includes the stepof administering to a patient a compound of formula I for a time and inan amount sufficient to treat the cryptic phase of the bacterialinfection. The chronic disease may be an inflammatory disease. Examplesof inflammatory diseases include, but are not limited to, asthma,coronary artery disease, arthritis, conjunctivitis, lymphogranulomavenerum (LGV), cervicitis, and salpingitis. The chronic disease can alsobe an autoimmune disease (e.g., systemic lupus erythematosus, diabetesmellitus, or graft versus host disease).

The invention also features a method for treating a patient diagnosed asbeing infected with a bacterium having a multiplying form and anon-multiplying form by administering to the patient (i) a compound offormula I and (ii) a second antibiotic that is effective against themultiplying form of the bacterium, wherein the two antibiotics areadministered in amounts and for a duration that, in combination,effectively treat the patient. In a related aspect, the inventionfeatures a method of treating a patient with a chronic diseaseassociated with a persistent bacterial infection by administering acompound of formula I.

In preferred embodiments of any of the foregoing aspects, the persistentintracellular bacterial infection is caused by one of the following:Chlamydia spp. (e.g., C. trachomatis, C. pneumoniae, C. psittaci, C.suis, C. pecorum, C. abortus, C. caviae, C. felis, C. muridarum), N.hartmannellae, W. chondrophila, S. negevensis, or P. acanthamoeba.

The time sufficient to treat a bacterial infection ranges from one weekto one year, but it can also be extended over the lifetime of theindividual patient, if necessary. In more preferable embodiments, theduration of treatment is at least 30 days, at least 45 days, at least100 days, or at least 180 days. Ultimately, it is most desirable toextend the treatment for such a time that the bacterial infection is nolonger detected.

Compounds of formula I are useful against drug resistant Gram-positivecocci, such as methicillin-resistant S. aureus and vancomycin-resistantenterococci, and are useful in the treatment of community-acquiredpneumonia, upper and lower respiratory tract infections, skin and softtissue infections, hospital-acquired lung infections, bone and jointinfections, and other bacterial infections.

The compounds and methods of the present invention can be used to treat,for example, respiratory tract infections, acute bacterial otitis media,bacterial pneumonia, urinary tract infections, complicated infections,noncomplicated infections, pyelonephritis, intra-abdominal infections,deep-seated abcesses, bacterial sepsis, skin and skin structureinfections, soft tissue infections, bone and joint infections, centralnervous system infections, bacteremia, wound infections, peritonitis,meningitis, infections after bum, urogenital tract infections,gastro-intestinal tract infections, pelvic inflammatory disease,endocarditis, and other intravascular infections.

The compounds and methods of the present invention can also be used totreat diseases associated with bacterial infection. For example,bacterial infections can produce inflammation, resulting in thepathogenesis of atherosclerosis, multiple sclerosis, rheumatoidarthritis, diabetes, Alzheimer's disease, asthma, cirrhosis of theliver, psoriasis, meningitis, cystic fibrosis, cancer, or osteoporosis.Accordingly, the present invention also features a method of treatingthe diseases associated with bacterial infection listed above.

The methods of the present invention can be used to treat or preventinfections by bacteria from a variety of genera, such as Escherichiaspp., Enterobacter spp., Enterobacteriaceae spp., Klebsiella spp.,Serratia spp., Pseudomonas spp., Acinetobacter spp., Bacillus spp.,Micrococcus spp., Arthrobacter spp., Peptostreptococcus spp.,Staphylococcus spp., Enterococcus spp., Streptococcus spp., Haemophilusspp., Neisseria spp., Bacteroides spp., Citrobacter spp., Branhamellaspp., Salmonella spp., Shigella spp., Proteus spp., Clostridium spp.,Erysipelothrix spp., Listeria spp., Pasteurella spp., Streptobacillusspp., Spirillum spp., Fusospirocheta spp., Treponema spp., Borreliaspp., Actinomycetes spp., Mycoplasma spp., Chlamydia spp., Rickettsiaspp., Spirochaeta spp., Legionella spp., Mycobacteria spp., Ureaplasmaspp., Streptomyces spp., and Trichomoras spp. Accordingly, the inventionfeatures a method of treating infections by the bacteria belonging tothe genera above, among others.

Particular Gram-positive bacterial infections that can be treatedaccording to the methods of the invention include infections byStaphylococcus aureus, Staphylococcus epidermidis, Enterococcusfaecalis,Enterococcusfaecium, Clostridium perfringens, Streptococcus pyogenes,Streptococcus pneumoniae, other Streptococcus spp., and otherClostridium spp.

Multi-drug resistant strains of bacteria can be treated according to themethods of the invention. Resistant strains of bacteria includepenicillin-resistant, methicillin-resistant, quinolone-resistant,macrolide-resistant, and/or vancomycin-resistant bacterial strains. Themulti-drug resistant bacterial infections to be treated using themethods of the present invention include infections by penicillin-,methicillin-, macrolide-, vancomycin-, and/or quinolone-resistantStreptococcus pneumoniae; penicillin-, methicillin-, macrolide-,vancomycin-, and/or quinolone-resistant Staphylococcus aureus;penicillin-, methicillin-, macrolide-, vancomycin-, and/orquinolone-resistant Streptococcus pyogenes; and penicillin-,methicillin-, macrolide-, vancomycin-, and/or quinolone-resistantenterococci.

The invention also features a method of eradicating non-multiplyingbacteria that are not eradicated in a patient following treatment with afirst antibiotic by administering to the patient a compound of formula Iin an amount and for a duration sufficient to eradicate thenon-multiplying bacteria in the patient.

Compounds of the invention may also be used to treat or prevent viralinfections.

In another aspect, the invention features a pharmaceutical compositionthat includes a compound described herein in any pharmaceuticallyacceptable form, including isomers such as diastereomers andenantiomers, salts, solvates, and polymorphs thereof. In variousembodiments, the composition includes a compound of the invention alongwith a pharmaceutically acceptable carrier or diluent.

In another aspect, the invention features a method of treating amicrobial infection in an animal comprising co-administering a compoundof the invention along with one or more antifungal agents, antiviralagents, antibacterial agents, or antiprotozoan agents, or combinationsthereof.

As used herein, the terms “alkyl” and the prefix “alk-” are inclusive ofboth straight chain and branched chain saturated or unsaturated groups,and of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Unlessotherwise specified, acyclic alkyl groups are from 1 to 6 carbons.Cyclic groups can be monocyclic or polycyclic and preferably have from 3to 8 ring carbon atoms. Exemplary cyclic groups include cyclopropyl,cyclopentyl, cyclohexyl, and adamantyl groups. Alkyl groups may besubstituted with one or more substituents or unsubstituted. Exemplarysubstituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio,halogen, alkylsilyl, hydroxyl, fluoroalkyl, perfluoralkyl, amino,aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl,carboxyalkyl, and carboxyl groups.

By “aryl” is meant a carbocyclic aromatic ring or ring system. Unlessotherwise specified, aryl groups are from 6 to 18 carbons. Examples ofaryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenylgroups.

By “heteroaryl” is meant an aromatic ring or ring system that containsat least one ring hetero-atom (e.g., O, S, Se, N, or P). Unlessotherwise specified, heteroaryl groups are from 1 to 9 carbons.Heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl,pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,tetrazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl, pyrimidyl,pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole,indazolyl, indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl,cinnolinyl, quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl,purinyl, and carbazolyl groups.

By “heterocycle” is meant a non-aromatic ring or ring system thatcontains at least one ring heteroatom (e.g., O, S, Se, N, or P). Unlessotherwise specified, heterocyclic groups are from 2 to 9 carbons.Heterocyclic groups include, for example, dihydropyrrolyl,tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl,tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophene,tetrahydrothiophene, and morpholinyl groups.

Aryl, heteroaryl, or heterocyclic groups may be unsubstituted orsubstituted by one or more substituents selected from the groupconsisting of C₁₋₆ alkyl, hydroxy, halo, nitro, C₁₋₆ alkoxy, C₁₋₆alkylthio, trifluoromethyl, C₁₋₆ acyl, arylcarbonyl, heteroarylcarbonyl,nitrile, C₁₋₆ alkoxycarbonyl, C₁₋₄ alkaryl and C₁₋₄ alkheteroaryl.

By “alkoxy” is meant a chemical substituent of formula —OR, where R isan alkyl group of 1 to 6 carbons, unless otherwise specified.

By “aryloxy” is meant a chemical substituent of formula —OR′, where R′is an aryl group of 6 to 18 carbons, unless otherwise specified.

By “C_(x-y) alkaryl” is meant a chemical substituent of formula —RR′,where R is an alkyl group of x to y carbons and R′ is an aryl group asdefined elsewhere herein.

By “C_(x-y) alkheteraryl” is meant a chemical substituent of formulaRR″, where R is an alkyl group of x to y carbons and R″ is a heteroarylgroup as defined elsewhere herein.

By “halide” or “halogen” or “halo” is meant bromine, chlorine, iodine,or fluorine.

By “non-vicinal O, S, or NR” is meant an oxygen, sulfur, or nitrogenheteroatom substituent in a linkage, where the heteroatom substituentdoes not form a bond to a saturated carbon that is bonded to anotherheteroatom.

In structural representations where the chirality of a carbon has beenleft unspecified, it is to be presumed by one skilled in the art thateither chiral form of that stereocenter is possible.

By “benzoxazinorifamycin” is meant a compound described by formula (A):

where W is O. By “benzthiazinorifamycin” is meant a compound describedby formula (A), where W is S. By “benzdiazinorifamycin” is meant acompound described by formula (A), where W is N—R. Forbenzdiazinorifamycin, R can be H or an alkyl substituent. When R is analkyl substituent, it is referred to as N′—R (e.g., N′-methyl) in thenaming of the compound. Benzoxazinorifamycin, benzthiazinorifamycin, andbenzdiazinorifamycin analogs that contain substituents are numberedaccording to the numbering provided in formula (A). By “25-O-deacetyl”rifamycin is meant a rifamycin analog in which the acetyl group at the25-position has been removed. Analogs in which this position is furtherderivatized are referred to as a“25-O-deacetyl-25-(substituent)rifamycin”,in which the nomenclature forthe derivatizing group replaces “substituent” in the complete compoundname. For example, a benzoxazinorifamycin analog in which the25-acetyloxy group has been transformed to a carbonate group, with theother side of the carbonate bonded to a 2,3-dihydroxypropyl group, isreferred to as a“25-O-deacetyl-25-(2″,3″-dihydroxypropylcarbonoxy)-benzoxazinorifamycin.”

By “atherosclerosis” is meant the progressive accumulation of smoothmuscle cells, immune cells (e.g., lymphocytes, macrophages, ormonocytes), lipid products (e.g., lipoproteins, or cholesterol),cellular waste products, calcium, or other substances within the innerlining of an artery, resulting in the narrowing or obstruction of theblood vessel and the development of atherosclerosis-associated diseases.Atherosclerosis is typically manifested within large and medium-sizedarteries, and is often characterized by a state of chronic inflammationwithin the arteries.

By “atherosclerosis-associated disease” is meant any disorder that iscaused by or is associated with atherosclerosis. Typically,atherosclerosis of the coronary arteries commonly causes coronary arterydisease, myocardial infarction, coronary thrombosis, and anginapectoris. Atherosclerosis of the arteries supplying the central nervoussystem frequently provokes strokes and transient cerebral ischemia. Inthe peripheral circulation, atherosclerosis causes intermittentclaudication and gangrene and can jeopardize limb viability.Atherosclerosis of an artery of the splanchnic circulation can causemesenteric ischemia. Atherosclerosis can also affect the kidneysdirectly (e.g., renal artery stenosis).

A patient who is being treated for an atherosclerosis-associated diseaseis one who a medical practitioner has diagnosed as having such adisease. Diagnosis may be by any suitable means. Methods for diagnosingatherosclerosis by measuring systemic inflammatory markers aredescribed, for example, in U.S. Pat. No. 6,040,147, hereby incorporatedby reference. Diagnosis and monitoring may employ an electrocardiogram,chest X-ray, echocardiogram, cardiac catheterization, ultrasound (forthe measurement of vessel wall thickness), or measurement of bloodlevels of CPK, CPK-MB, myoglobin, troponin, homocysteine, or C-reactiveprotein. A patient in whom the development of anatherosclerosis-associated disease is being prevented is one who has notreceived such a diagnosis. One in the art will understand that thesepatients may have been subjected to the same tests (electrocardiogram,chest X-ray, etc.) or may have been identified, without examination, asone at high risk due to the presence of one or more risk factors (e.g.,family history, hypertension, diabetes mellitus, high cholesterollevels). Thus, prophylactic administration of a rifamycin analog isconsidered to be preventing the development of anatherosclerosis-associated disease.

An atherosclerosis-associated disease has been treated or prevented whenone or more tests of the disease (e.g., any of the those describedabove) indicate that the patient's condition has improved or thepatient's risk reduced. In one example, a reduction in C-reactiveprotein to normal levels indicates that an atherosclerosis-associateddisease has been treated or prevented.

An alternative means by which treatment or prevention is assessedincludes determination of the presence of an infection of C. pneumoniae.Any suitable method may be employed (e.g., determination of C.pneumoniae in blood monocytes or in the atheroma itself (e.g., inmacrophages or foam cells present in the fatty streak), or detection ofC. pneumoniae DNA, RNA, or antibodies to C. pneumoniae in a biologicalsample from the patient).

By “debris” is meant the mucoid exudate or desquamated epithelium in aninfected ear of a patient having an ear infection.

By “ear wick” is meant a sponge, cotton, gauze, compressedhydroxycellulose, or any other material used to increase the penetrationof rifamycin to the infected otic area. The ear wick is typicallyinserted into the canal under direct vision. Its presence helps wickeardrops along the canal, hold the solution in contact with the skin ofthe canal, and apply pressure to the canal skin.

By “granulation tissue” is meant the highly vascularized tissue thatreplaces the initial fibrin clot in a wound. Vascularization is a resultof an ingrowth of capillary endothelium from the surroundingvasculature. The tissue is also rich in fibroblasts and leucocytes.

“Antibiotic-associated bacterial diarrhea” means the condition whereinantibiotic therapy disturbs the balance of the microbial flora of thegut, allowing pathogenic organisms such as C. difficile to flourish.These organisms cause diarrhea. Antibiotic-associated bacterial diarrheaincludes such conditions as C. difficile associated diarrhea (CDAD) andpseudomembranous colitis.

“Pseudomembranous colitis,” also known as pseudomembranous enterocolitisor enteritis, means the inflammation of the mucous membrane of bothsmall and large intestine with the formation and passage ofpseudomembranous material (composed of fibrin, mucous, necroticepithelial cells and leukocytes) in the stools.

By “autoimmune disease” is meant a disease arising from an immunereaction against self-antigens and directed against the individual's owntissues. Examples of autoimmune diseases include but are not limited tosystemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis,and Graves' disease.

By “bacteria” is meant a unicellular prokaryotic microorganism thatusually multiplies by cell division.

By “bacterial infection” is meant the invasion of a host animal bypathogenic bacteria. For example, the infection may include theexcessive growth of bacteria that are normally present in or on the bodyof an animal or growth of bacteria that are not normally present in oron the animal. More generally, a bacterial infection can be anysituation in which the presence of a bacterial population(s) is damagingto a host animal. Thus, an animal is “suffering” from a bacterialinfection when an excessive amount of a bacterial population is presentin or on the animal's body, or when the presence of a bacterialpopulation(s) is damaging the cells or other tissue of the animal.

By “chronic disease” is meant a disease that is inveterate, of longcontinuance, or progresses slowly, in contrast to an acute disease,which rapidly terminates. A chronic disease may begin with a rapid onsetor in a slow, insidious manner but it tends to persist for severalweeks, months or years, and has a vague and indefinite termination.

By “immunocompromised” is meant a person who exhibits an attenuated orreduced ability to mount a normal cellular or humoral defense tochallenge by infectious agents, e.g., viruses, bacterial, fungi, andprotozoa. Persons considered immunocompromised include malnourishedpatients, patients undergoing surgery and bone narrow transplants,patients undergoing chemotherapy or radiotherapy, neutropenic patients,HIV-infected patients, trauma patients, burn patients, patients withchronic or resistant infections such as those resulting frommyelodysplastic syndrome, and the elderly, all of who may have weakenedimmune systems.

By “inflammatory disease” is meant a disease state characterized by (1)alterations in vascular caliber that lead to an increase in blood flow,(2) structural changes in the microvasculature that permit the plasmaproteins and leukocytes to leave the circulation, and (3) emigration ofthe leukocytes from the microcirculation and their accumulation in thefocus of injury. The classic signs of acute inflammation are erythema,edema, tenderness (hyperalgesia), and pain. Chronic inflammatorydiseases are characterized by infiltration with mononuclear cells (e.g.,macrophages, lymphocytes, and plasma cells), tissue destruction, andfibrosis. Non-limiting examples of inflammatory disease include asthma,coronary artery disease, arthritis, conjunctivitis, lymphogranulomavenerum, and salpingitis.

By “intracytoplasmic inclusion” is meant a replicating reticulate body(RB) that has no cell wall. Such inclusions may be detected, forexample, through chlamydiae sample isolation and propagation onmammalian cell lines, followed by fixing and staining using one of avariety of staining methods including Giemsa staining, iodine staining,and immunofluorescence. These inclusions have a typical round or ovalappearance.

By “persistent bacterial infection” is meant an infection that is notcompletely eradicated through standard treatment regimens usinganti-bacterial agents. Persistent bacterial infections are caused bybacteria capable of establishing a cryptic or latent phase of infectionand may be classified as such by culturing the bacteria from a patientand demonstrating bacterial survival in vitro in the presence ofanti-bacterial agents or by determination of anti-bacterial treatmentfailure in a patient. As used herein, a persistent infection in apatient includes any recurrence of chlamydial infection, after receivinganti-bacterial treatment, from the same species (e.g., C. trachomatis)more than two times over the period of two or more years or thedetection of the cryptic phase of the infection in the patient by themethods described. An in vivo persistent infection can be identifiedthrough the use of a reverse transcriptase polymerase chain reaction(RT-PCR) to demonstrate the presence of 16S rRNA transcripts inbacterially infected cells after treatment with anti-bacterial agents(Antimicrob. Agents Chemother. 12:3288-3297, 2000).

By “replicating phase” is meant the phase of the bacterial cell cyclecharacterized by the presence of an RB. The RB is the activelyreplicating form of the Chlamydia. It contains no cell wall and isdetected as an inclusion in the cell.

As used herein, the term “treating” refers to administering orprescribing a pharmaceutical composition for prophylactic and/ortherapeutic purposes. To “prevent disease” refers to prophylactictreatment of a patient who is not yet ill, but who is susceptible to, orotherwise at risk of, a particular disease. To “treat disease” or usefor “therapeutic treatment” refers to administering treatment to apatient already suffering from a disease to improve the patient'scondition. Thus, in the claims and embodiments, treating is theadministration to an animal either for therapeutic or prophylacticpurposes. An ear infection has been treated when one or more tests ofthe disease (e.g., any of the those described below) indicate that thepatient's condition has improved. The detection of an infection may bedone by a pneumatic otoscopic examination of the patient, or by areduction in infection-associated symptoms in the patient (e.g.,inflammation of ear drums, redness of ear drums, presence of fluid inears). Reduction of symptoms may also be determined, for example, byaudiogram to check recovery from hearing loss. Prophylacticadministration of a rifamycin of the invention is considered to bepreventing the development of an ear infection.

By “effective amount” is meant the amount of a compound required totreat or prevent an infection. The effective amount of activecompound(s) used to practice the present invention for therapeutic orprophylactic treatment of conditions caused by or contributed to by amicrobial infection varies depending upon the manner of administration,the age, body weight, and general health of the subject. Ultimately, theattending physician or veterinarian will decide the appropriate amountand dosage regimen. Such amount is referred to as an “effective” amount.

The term “microbial infection” refers to the invasion of the host animalby pathogenic microbes. This includes the excessive growth of microbesthat are normally present in or on the body of an animal. Moregenerally, a microbial infection can be any situation in which thepresence of a microbial population(s) is damaging to a host animal.Thus, an animal is “suffering” from a microbial infection when excessivenumbers of a microbial population are present in or on an animal's body,or when the presence of a microbial population(s) is damaging the cellsor other tissue of an animal.

The term “microbes” includes, for example, bacteria, fungi, yeasts,viruses and protozoa.

By “intracellular pathogen” is meant an infection by any facultative orobligate intracellular microbe.

By “obligate intracellular pathogen” is meant a microbe that must use anintracellular location (e.g., a host cell) in order to replicate.

By “facultative intracellular pathogen” is meant a microbe that is ableto survive within an intracellular location (e.g., a host cell), butdoes not require an intracellular environment to replicate.

The term “administration” or “administering” refers to a method ofgiving a dosage of a pharmaceutical composition to an animal, where themethod is, e.g., topical, oral, intravenous, intraperitoneal, orintramuscular. The preferred method of administration can vary dependingon various factors, e.g., the components of the pharmaceuticalcomposition, site of the potential or actual disease, and severity ofdisease.

The terms “animal,” “subject,” and “patient” specifically includehumans, cattle, horses, dogs, cats, and birds, but also can include manyother species.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered rifamycin analogs that are useful for treating orpreventing a variety of microbial infections. The compounds of thepresent invention can be described by formula I:

or a pharmaceutically acceptable salt thereof,wherein

A is H, OH, O—(C₁₋₆ alkyl), O—(C₁₋₄ alkaryl), O—(C₆₋₁₂ aryl), O—(C₁₋₉heteroaryl), or O—(C₁₋₄ alkheteroaryl);

W is O, S, or NR¹, wherein R¹ is H, C₁₋₆ alkyl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl;

X is H or COR², wherein R² is C₁₋₆ alkyl, which can be substituted with1-5 OH groups, O—(C₃₋₇ alkyl), which can be substituted with 1-4 OHgroups, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄alkheteroaryl, wherein each alkyl carbon is bonded to to no more thanone oxygen atom;

Y is H, Hal, or OR^(Y3), wherein R^(Y3) is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl;

Z is H, Hal, or OR^(Z3), wherein R^(Z3) is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl; and

R⁴ has the formula:

wherein

R⁵ is H, C₁₋₆ alkyl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, COR¹⁰, CO₂R¹¹,CONR¹⁰R¹¹ CSR¹⁰, COSR¹¹, CSOR¹¹, CSNR¹⁰OR¹¹, SO₂R¹¹, or SO₂NR¹⁰R¹¹,wherein R¹⁰ is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl,or C₁₋₄ alkheteroaryl, R¹¹ is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉heteroaryl, or C₁₋₄ alkheteroaryl, or R¹⁰ and R¹¹ together form a C₂₋₆linkage, optionally containing a non-vicinal O;

R6 is H, C₁₋₆ alkyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl;

R⁷ is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₉ heteroaryl, C₂₋₉ heterocyclyl,C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, OR¹², or NR¹²R¹³, wherein R¹² is H,C₆₋₁₂ alkyl, C₁₋₆ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄alkheteroaryl, R¹³ is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉heteroaryl, or C₁₋₄ alkheteroaryl, or R¹² and R¹³ together form a C₂₋₆linkage, optionally containing a non-vicinal O;

T is O, S, NR⁵, or a bond;

each of R⁸ and R⁹ is, independently H, C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₉heteroaryl, C₂₋₉ heterocyclyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, orR⁸ and R⁵ together form a 3-8-membered ring, with the ring optionallycontaining a non-vicinal oxygen; and each of r and s is, independently,1 or 2.

We have identified a method of preventing, stabilizing, or inhibitingthe growth of microbes, or killing microbes. The method involvescontacting microbes or a site susceptible to microbial growth with acompound of the invention. Compounds of the present invention can beused to treat, stabilize or prevent a microbial infection in an animal.In this method, the step of contacting microbes or a site susceptible tomicrobial infection (e.g., a site in or on the body of an animal) with acompound of the invention includes administering to the animal thecompound in an amount sufficient to treat, stabilize, or prevent themicrobial infection in the animal. In a related aspect, the inventionfeatures a method of treating any disease associated with such amicrobial infection.

Compounds of the present invention can be used to treat atherosclerosisor diseases associated therewith, sexually transmitted diseases caused,for example, by C. trachomatis or N. gonorrhoeae, otitis media and otherear infections, antibiotic-associated colitis, gastritis and ulcersassociated with an infection of H. pylori, Gram-positive infections,community-acquired pneumonia, upper and lower respiratory tractinfections, skin and soft tissue infections, bone and joint infections,hospital-acquired lung infections, urinary tract infections,pyelonephritis, intra-abdominal infections, bacteremia, bacterialsepsis, would infections, peritonitis, osteomyelitis, infections afterbums, pelvic inflammatory disease, and diseases associated with chronicinfections.

Atherosclerosis and other Diseases Associated with Chlamydial Infection

An association was previously reported in International Publication No.WO 98/50074 between the cryptic phase of a persistent chlamydialinfection of body fluids and/or tissues and several chronic diseasesyndromes of previously unknown etiology in humans. To date, thesediseases include, but are not limited to, atherosclerosis, multiplesclerosis, rheumatoid arthritis, inflammatory bowel disease,interstitial cystitis, fibromyalgia, autonomic nervous dysfunction(neural-mediated hypotension); pyoderma gangrenosum, and chronic fatiguesyndrome.

As described in International Publication No. WO 98/50074, several linesof evidence have led to the establishment of a link between Chlamydiaand a broad set of inflammatory, autoimmune, and immune deficiencydiseases. These include (i) the association between the cryptic phase ofa persistent chlamydial infection of body fluids and/or tissues andseveral chronic disease syndromes as described above, (ii) publishedevidence of an association between atherosclerosis and Chlamydia(Circulation, 96:404-407, 1997), and (iii) an understanding of theimpact the persistent infection established by the cryptic phase ofchlamydial infections can have on infected cells and the immune system.Thus, the present invention describes methods for treating chronicdiseases associated with the cryptic phase of a persistent chlamydialinfection, such as autoimmune diseases, inflammatory diseases anddiseases that occur in immunocompromised individuals by treating thecryptic phase of the infection in an individual in need thereof, usingthe rifamycin analogs described herein. Progress of the treatment can beevaluated, using the diagnostic tests described herein, to determine thepresence or absence of Chlamydia. Physical improvement in the conditionsand symptoms typically associated with the disease to be treated canalso be evaluated. Based upon these evaluating factors, the physiciancan maintain or modify the anti-bacterial therapy accordingly.

The therapies described herein can be used for the treatment of chronicimmune and autoimmune diseases when patients are demonstrated to have aChlamydia load by the methods of detection described above. Thesediseases include, but are not limited to, chronic hepatitis, systemiclupus erythematosus, arthritis, thyroidosis, scleroderma, diabetesmellitus, Graves' disease, Behcet's disease, and graft versus hostdisease (graft rejection). The therapies of this invention can also beused to treat any disorders in which a chlamydial species is a factor orco-factor.

Thus, the present invention can be used to treat a range of disorders inaddition to the above immune and autoimmune diseases when demonstratedto be associated with chlamydial infection by the methods of detectiondescribed herein; for example, various infections, many of which produceinflammation as primary or secondary symptoms, including, but notlimited to, sepsis syndrome, cachexia, circulatory collapse and shockresulting from acute or chronic bacterial infection, acute and chronicparasitic and/or infectious diseases from bacterial, viral or fungalsources, such as a HIV, AIDS (including symptoms of cachexia, autoimmunedisorders, AIDS dementia complex and infections) can be treated, as wellas Wegners Granulomatosis.

Among the various inflammatory diseases, there are certain features thatare generally agreed to be characteristic of the inflammatory process.These include fenestration of the microvasculature, leakage of theelements of blood into the interstitial spaces, and migration ofleukocytes into the inflamed tissue. On a macroscopic level, this isusually accompanied by the familiar clinical signs of erythema, edema,tenderness (hyperalgesia), and pain. Inflammatory diseases, such aschronic inflammatory pathologies and vascular inflammatory pathologies,including chronic inflammatory pathologies such as aneurysms,hemorrhoids, sarcoidosis, chronic inflammatory bowel disease, ulcerativecolitis, and Crohn's disease and vascular inflammatory pathologies, suchas, but not limited to, disseminated intravascular coagulation,atherosclerosis, and Kawasaki's pathology are also suitable fortreatment by methods described herein. The invention can also be used totreat inflammatory diseases such as coronary artery disease,hypertension, stroke, asthma, chronic hepatitis, multiple sclerosis,peripheral neuropathy, chronic or recurrent sore throat, laryngitis,tracheobronchitis, chronic vascular headaches (including migraines,cluster headaches and tension headaches), and pneumonia.

Treatable disorders when associated with a chlamydial infection alsoinclude, but are not limited to, neurodegenerative diseases, including,but not limited to, demyelinating diseases, such as multiple sclerosisand acute transverse myelitis; extrapyramidal and cerebellar disorders,such as lesions of the corticospinal system; disorders of the basalganglia or cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; progressivesupranucleo palsy; cerebellar and spinocerebellar disorders, such asastructural lesions of the cerebellum; spinocerebellar degenerations(spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations,multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, andMachado Joseph)); and systemic disorders (Refsum's disease,abetalipoprotemia, ataxia, telangiectasia, and mitochondrialmulti-system disorder); demyelinating core disorders, such as multiplesclerosis, acute transverse myelitis; disorders of the motor unit, suchas neurogenic muscular atrophies (anterior horn cell degeneration, suchas amyotrophic lateral sclerosis, infantile spinal muscular atrophy andjuvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndromein middle age; Diffuse Lewy body disease; senile dementia of Lewy bodytype; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakobdisease; subacute sclerosing panencephalitis, Hallerrorden-Spatzdisease; and Dementia pugilistica, or any subset thereof.

It is also recognized that malignant pathologies involving tumors orother malignancies, such as, but not limited to leukemias (acute,chronic myelocytic, chronic lymphocytic and/or myelodyspastic syndrome);lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such as malignantlymphomas (Burkitt's lymphoma or mycosis fungoides)); carcinomas (suchas colon carcinoma) and metastases thereof; cancer-related angiogenesis;infantile hemangiomas; and alcohol-induced hepatitis. Ocularneovascularization, psoriasis, duodenal ulcers, and angiogenesis of thefemale reproductive tract can also be treated when demonstrated by thediagnostic procedures described herein to be associated with achlamydial infection.

Ear Infections

Ear infections typically affect the middle or the external ear andinclude, for example, otitis media, otitis externa, and infectionscaused by surgical interventions. Due to multiplicity of secondarycomplications that arise from ear infections such as hearing loss, thetreatment and prevention of such conditions is critical.

Topical administration of a compound of formula I is effective intreating or preventing an infection of the ear, such as otitis media orotitis extema. In the case of otitis media or externa, infections areprimarily caused by H. influenza, M. catarhalis, S. pneumoniae, S.pyogenes, S. intermedius, S. epidermidis, S. aureus, S. caprae, S.auriculis, S. capitis, S. haemolytis, P. aeroginosa, P. mirabilis, P.vulgaris, E. faecalis, or E. coli. A compound of formula I can be usedto treat each of these infections of the ear. A compound of formula Imay, for example, be topically administered to the area of the ear towhich surgical intervention was performed or, alternatively, thecompound may be administered to the ear of the patient prophylactically,prior to otic surgery, noninvasive otic procedures, or other types ofsurgery. Exemplary surgical procedures include for example, cochlearimplant surgery, tympanoplasty, tympanostomy tube insertion, removal oftumors (e.g., cholesteatoma), or stapedectomy. The compound may beadministered to the area of the ear to which surgical intervention willbe performed, for example, within seven days, two days, one day, 12hours, 10 hours, 6 hours, 4 hours, 2 hours, 1 hour, or less than 1 hourprior to or following the surgical intervention. The compositions may beused for acute treatment of temporary conditions, or may be administeredchronically.

A compound of formula I may be given daily (e.g., once, twice, threetimes, or four times daily) or less frequently (e.g., once every otherday, or once or twice weekly). Typically, patients are administered adosage consisting of one to four drops of solution containing thecompound. The compound may be contained in any appropriate amount in anysuitable carrier substance, and is generally present in an amountbetween 0.001% and 5%, desirably 0.01% and 3%, more desirably 0.1% and1%, and even more desirably 0.1% and 0.4% by weight of the total volume(w/v) of the composition. The compound is provided in a dosage form thatis suitable for topical administration. Thus, a composition containing acompound of formula I may be in the form of a solution, aerosol, gel,ointment, nebulizer, or suspension. Alternatively, a compound of theinvention may be administered by placing an impregnated porous mediainto the external ear canal to the tympanic membrane. The pharmaceuticalcomposition can generally be formulated according to conventionalpharmaceutical practice.

Aural Toilet

The external auditory canal and tissues lateral to the infected middleear often are covered with mucoid exudate or desquamated epithelium.Since topically applied preparations cannot generally penetrate affectedtissues until these interposing materials are removed, aural toilet isdesirably performed before administering a compound of formula I. Auraltoilet may be performed by a health provider, the patient, or any otherindividual. Removal of debris may be performed mechanically with theassistance of a microscope and microinstruments. Aural irrigation mayalso be performed using a solution containing peroxide. Theconcentration of peroxide should be the highest concentration withoutcausing significant pain, or discomfort, to the patient. As an example,a solution of 50% peroxide and 50% sterile water can be used. Thirty to40 mL of this solution can be irrigated through the external auditorycanal, using a small syringe or bulb-type aspirator. The irrigantsolution is allowed to drain out (e.g., for 5-10 minutes) prior toadministering a compound of the invention.

Granulation Tissue

Granulation tissue often fills the middle ear and medial portions of theexternal auditory canal, and reducing this accumulation is beneficialfor resolution of an ear infection. Granulation tissue may also preventtopically applied antimicrobial agents from penetrating to the site ofinfection, and the amount of granulation tissue is desirably reducedthroughout the regimen.

Although topical antimicrobial drops can reduce granulation byeliminating infection and by removing the inciting irritatinginflammation, the amount of granulation tissue may be reduced usingother methods known in the art. For example, topical steroids may hastenthe resolution of middle ear granulation, thus improving penetration oftopically delivered antibiotics.

Cautery may also be used to reduce the amount of granulation tissue andto reduce its formation. Microbipolar cautery may be administered by ahealth provider. Chemical cautery, using for example silver nitrate, mayalso be applied to an infected ear in the form of silver nitrate sticks.Excision of granulation tissue may also be performed by a health careprovider with a microscope and microinstruments.

Ear Canal Acidification

In a patient affected with otitis externa, a therapy involving ear canalacidification to restore the physiological acidity of the ear may beperformed. The affected ear is administered with a solution containingacetic acid, which may also include a steroid (e.g., hydrocortisone),aluminum acetate, or rubbing alcohol.

Topical Formulations

Pharmaceutical compositions according to the present invention can beformulated for topical administration to the ear of the patient.Patients having an ear infection may be administered with effectiveamounts of a compound of the invention, by means of a solution (e.g.,drops), ointment, gel, or aerosol (e.g., nebulizer). The composition istypically administered to the affected otic area by topically applying,for example, one to four drops of a solution or suspension, or acomparable amount of an ointment, gel, or other solid or semisolidcomposition, once, twice, three times, or more than three times per day.A porous media or an ear wick (e.g., cotton, gauze, or compressedhydroxycellulose) may also be used to increase the penetration of acompound of the invention to the infected otic area. The ear wick, whichis inserted into the canal under direct vision, is typically a driedsponge that helps wick eardrops along the canal, hold the solution incontact with the skin of the canal and apply pressure to the canal skin.Wicks may be removed at one day, two days, or more than two days, andmay be replaced if necessary. Alternatively, the ear wick may itself beimpregnated with a compound of the invention. These formulations can bemade according to known and conventional methods for preparing suchformulations.

For compounds of the invention that are not highly soluble in water atphysiological conditions, a solubilizing excipient may be used toincrease solubility. Solubilization is taken to mean an improvement inthe solubility by virtue of surface-active compounds that can convertsubstances that are insoluble or virtually insoluble in water intoclear, or opalescent, aqueous solutions without changing the chemicalstructure of these substances in the process. Excipients used for thispurpose are restricted to those that are safe for administration tohumans. Typically such co-solvents are employed at a level of about0.01% to 2% by weight.

A variety of solubilizing excipients may be used for the formulation ofa compound of the invention, including compounds belonging to thefollowing classes: polyethoxylated fatty acids, PEG-fatty acid diesters,PEG-fatty acid mono-ester and di-ester mixtures, polyethylene glycolglycerol fatty acid esters, alcohol-oil transesterification products,polyglycerized fatty acids, propylene glycol fatty acid esters, mixturesof propylene glycol esters and glycerol esters, mono- and diglycerides,sterol and sterol derivatives, polyethylene glycol sorbitan fatty acidesters, polyethylene glycol alkyl ethers, sugar esters, polyethyleneglycol alkyl phenols, polyoxyethylene-polyoxypropylene block copolymers,sorbitan fatty acid esters, lower alcohol fatty acid esters, or ionicsurfactants. Such excipients are described for example, in U.S. patentapplication Ser. No. 60/385,532, hereby incorporated by reference.

Ototopical preparations may vary in viscosity. The use of viscosityenhancing agents to provide the compositions of the invention withviscosities greater than the viscosity of simple aqueous solutions maybe desirable to increase the retention time in the ear. Suchviscosity-building agents include, for example, polyvinyl alcohol,polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropylcellulose, or other agents known to those skilled in the art. Suchagents are typically employed at a level of about 0.01% to 2% by weight.Optionally, these preparations may include a buffering agent to maintainan acidic pH, since the normal environment of the external auditorycanal is acidic. However, if treatment is required in the middle earwhere the pH is neutral, the pH can be adjusted accordingly.

Otic pharmaceutical products are typically packaged in multidose form.Preservatives are thus desired to prevent microbial contamination duringuse. Suitable preservatives include: polyquatemium-1, benzalkoniumchloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben,phenylethyl alcohol, edetate disodium, sorbic acid, or other agentsknown to those skilled in the art. Typically such preservatives areemployed at a level of from 0.001% to 1.0% by weight.

A penetration enhancer may also be used to facilitate the diffusion of acompound of the invention through the tympanic membrane into the middleand inner ear in order to reduce inflammation of ear tissues. Apenetration enhancer is an agent used to increase the permeability ofthe skin to a pharmacologically active agent to increase the rate atwhich the drug diffuses through the skin and enters the tissues andbloodstream. A chemical skin penetration enhancer increases skinpermeability by reversibly damaging or by altering the physiochemicalnature of the stratum corneum to reduce its diffusional resistance(Osborne D W, Henke J J, Pharmaceutical Technology, November 1997, pp58-86). Examples of penetration enhancers include without limitation:alcohols, such as ethanol and isopropanol; polyols, such as n-alkanols,limonene, terpenes, dioxolane, propylene glycol, ethylene glycol, otherglycols, and glycerol; sulfoxides, such as dimethylsulfoxide (DMSO),dimethylformamide, methyl dodecyl sulfoxide, dimethylacetamide; esters,such as isopropyl myristate/palmitate, ethyl acetate, butyl acetate,methyl proprionate, and capric/caprylic triglycerides; ketones; amides,such as acetamides; oleates, such as triolein; various surfactants, suchas sodium lauryl sulfate; various alkanoic acids, such as caprylic acid;lactam compounds, such as azone; alkanols, such as oleyl alcohol;dialkylamino acetates, and admixtures thereof. The use of suchpenetration enhancers is disclosed, for example, in U.S. Pat. No.6,093,417, hereby incorporated by reference.

Other Therapeutic Agents

Compositions containing a compound of the invention may also include asecond therapeutic agent, such as, for example, another rifamycinanalog, an anesthetic, an antimicrobial agent, a zinc salt, or ananti-inflammatory agent (e.g., an non-steroidal anti-inflammatory or asteroid). When admixing an antimicrobial agent, the antimicrobial agentis preferably penicillin G, penicillin V, methicillin, oxacillin,cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin,carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin,temocillin, cepalothin, cephapirin, cephradine, cephaloridine,cefazolin, cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor,loracarbef, cefoxitin, cefmatozole, cefotaxime, ceftizoxime,ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime,ceftibuten, cefdinir, cefpirome, cefepime, BAL5788, BAL9141, imipenem,ertapenem, meropenem, astreonam, clavulanate, sulbactam, tazobactam,streptomycin, neomycin, kanamycin, paromycin, gentamicin, tobramycin,amikacin, netilmicin, spectinomycin, sisomicin, dibekalin, isepamicin,tetracycline, chlortetracycline, demeclocycline, minocycline,oxytetracycline, methacycline, doxycycline, erythromycin, azithromycin,clarithromycin, telithromycin, ABT-773, lincomycin, clindamycin,vancomycin, oritavancin, dalbavancin, teicoplanin, quinupristin anddalfopristin, sulphanilamide, para-aminobenzoic acid, sulfadiazine,sulfisoxazole, sulfamethoxazole, sulfathalidine, linezolid, nalidixicacid, oxolinic acid, norfloxacin, perfloxacin, enoxacin, ofloxacin,ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin,sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin,gemifloxacin, sitafloxacin, metronidazole, daptomycin, garenoxacin,ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, or trimethoprim.Preferred non-steroidal anti-inflammatory agents include, for example,detoprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, mechlofenameate, mefenamic acid, meloxicam,nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmeting,celecoxib, rofecoxib, choline salicylate, salsate, sodium salicylate,magnesium salicylate, aspirin, ibuprofen, paracetamol, acetaminophen,and pseudoephedrine, and preferred steroids include, for example,hydrocortisone, prednisone, fluprednisolone, triamcinolone,dexamethasone, betamethasone, cortisone, prednilosone,methylprednisolone, fluocinolone acetonide, flurandrenolone acetonide,and fluorometholone. Preferred anesthetics according to the inventioninclude, for example, benzocaine, butamben picrate, tetracaine,dibucaine, prilocaine, etidocaine, mepivacaine, bupivicaine, andlidocaine. A zinc salt can be zinc sulfate, zinc chloride, zinc acetate,zinc phenol sulfonate, zinc borate, zinc bromide, zinc nitrate, zincglycerophosphate, zinc benzoate, zinc carbonate, zinc citrate, zinchexafluorosilicate, zinc diacetate trihydrate, zinc oxide, zincperoxide, zinc salicylate, zinc silicate, zinc stannate, zinc tannate,zinc titanate, zinc tetrafluoroborate, zinc gluconate, or zincglycinate. All of the therapeutic agents employed in the compositions ofthe present invention can be used in the dose ranges currently known andused for these agents.

Different concentrations may be employed depending on the clinicalcondition of the patient, the goal of therapy (treatment orprophylaxis), the anticipated duration, and the severity of theinfection for which a compound of the invention is being administered.Additional considerations in dose selection include the type ofinfection, age of the patient (e.g., pediatric, adult, or geriatric),general health, and comorbidity.

Synthesis

Rifamycin analogs of formula I can be synthesized by methods analogousto those disclosed in U.S. Pat. Nos. 4,610,919; 4,983,602; 5,786,349;5,981,522; and 4,859,661, and Chem. Pharm. Bull., 41:148 (1993), each ofwhich is hereby incorporated by reference. Syntheses of rifamycinanalogs of formula I are provided in the Examples. The synthesis of the25-hydroxy prodrug analog (see Seligson, et al., Anti-Cancer Drugs12:305-13, 2001), 25-O-deacetyl-25-(2″,3″-dihydroxypropylcarbonoxy)-4′-fluoro-5′-[4-isobutyl-1-piperazinyl]benzoxazinorifamycin (Example 5and FIG. 1) can be used as a guide for the synthesis of other rifamycinanalogs in which the 25-hydroxyl group is derivatized (e.g., as anester, a carbamate, or a carbonate).

Assays

Compounds of the present invention can be screened for antimicrobialactivity by measuring their minimum inhibitory concentration (MIC),using standard MIC in vitro assays (see, for example, Tomioka et al.,Antimicrob. Agents Chemother. 37:67 (1993). Agents can be screenedagainst C. pneumoniae, C. trachomatis, M. tuberculosis (includingmultiple drug resistant strains), M. avium complex, and otherintracellular infectious microbes. Details of a standard MIC assay areprovided in Example 16.

Therapy

The invention features a method of treating or preventing a disease orcondition associated with a microbial infection by administering acompound of formulas (I). Compounds of the present invention may beadministered by any appropriate route for treatment or prevention of adisease or condition associated with a microbial infection,inflammation, or infection derived autoimmune disease. These may beadministered to humans, domestic pets, livestock, or other animals witha pharmaceutically acceptable diluent, carrier, or excipient, in unitdosage form. Administration may be topical, parenteral, intravenous,intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intracapsular, intraspinal, intracistemal,intraperitoneal, intranasal, aerosol, by suppositories, or oraladministration.

Therapeutic formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in Remington: The Science and Practice of Pharmacy (20th ed.,ed. A. R. Gennaro A R.), Lippincott Williams & Wilkins, 2000.Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel. Theconcentration of the compound in the formulation will vary dependingupon a number of factors, including the dosage of the drug to beadministered, and the route of administration.

The compound may be optionally administered as a pharmaceuticallyacceptable salt, such as a non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like.

Administration of compounds in controlled release formulations is usefulwhere the compound of formula I, has (i) a narrow therapeutic index(e.g., the difference between the plasma concentration leading toharmful side effects or toxic reactions and the plasma concentrationleading to a therapeutic effect is small; generally, the therapeuticindex, TI, is defined as the ratio of median lethal dose (LD₅₀) tomedian effective dose (ED₅₀)); (ii) a narrow absorption window in thegastro-intestinal tract; or (iii) a short biological half-life, so thatfrequent dosing during a day is required in order to sustain the plasmalevel at a therapeutic level.

Many strategies can be pursued to obtain controlled release in which therate of release outweighs the rate of metabolism of the therapeuticcompound. For example, controlled release can be obtained by theappropriate selection of formulation parameters and ingredients (e.g.,appropriate controlled release compositions and coatings). Examplesinclude single or multiple unit tablet or capsule compositions, oilsolutions, suspensions, emulsions, microcapsules, microspheres,nanoparticles, patches, and liposomes.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

Pharmaceutical formulations of compounds of the invention describedherein include isomers such as diastereomers and enantiomers, mixturesof isomers, including racemic mixtures, salts, solvates, and polymorphsthereof.

Compounds of the invention may be used in combination with anotherantifungal, antiviral, antibacterial, or antiprotozoan compound orcombinations thereof. The other agent may be employed as a supplementalantimicrobial for the purpose of broadening the activity spectrum of thetherapeutics or to obtain particular effects, such as, to reduce thedevelopment of drug resistant microbes. Compounds of the invention canbe used either alone or in conjunction with other pharmaceuticalcompounds to effectively combat a single infection. For example,compounds of the invention may be used either alone or combined withacyclovir in a combination therapy to treat HSV-1. Compounds of theinvention may also be used either alone or in conjunction with otherpharmaceutical compounds to combat multiple infections. For example,compounds of the invention may be used in combination with one or moreanti-mycobacterial agents agents such as isoniazid, pyrazinamide, orethambutol to treat or prevent intracellular bacterial infections.Compounds of the invention may also be used in combination with Intron Aand/or a biflavanoid for treating Hepatitis B; with gancyclovir,progancyclovir, famcyclovir, foscarnet, vidarabine, cidovir, and/oracyclovir for treating herpes viruses; and with ribavarin, amantidine,and/or rimantidine for treating respiratory viruses.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how themethods and compounds claimed herein are performed, made, and evaluated,and are intended to be purely exemplary of the invention and are notintended to limit the scope of what the inventors regard as theirinvention.

Syntheses of Benzoxazinorifamycin Compounds

Benzoxazinorifamycin compounds can be prepared using methods thatrequire the selective protection and deprotection of alcohols, amines,sulfhydryls and/or carboxylic acid functional groups. For example,commonly used protecting groups for amines include carbamates, such astert-butyl, benzyl, 2,2,2 -trichloroethyl, 2-trimethylsilylethyl,9-fluorenylmethyl, allyl, and m-nitrophenyl. Other commonly usedprotecting groups for amines include amides, such as formamides,acetamides, trifluoroacetamides, sulfonamides, trifluoromethanesulfonylamides, trimethylsilylethanesulfonamides, and tert-butylsulfonyl amides.Examples of commonly used protecting groups for carboxylic acids includeesters, such as methyl, ethyl, tert-butyl, 9-fluorenylmethyl,2-(trimethylsilyl)ethoxy methyl, benzyl, diphenylmethyl, O-nitrobenzyl,ortho-esters, and halo-esters. Examples of commonly used protectinggroups for alcohols include ethers, such as methyl, methoxymethyl,methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl,tetrahydropyranyl, ethoxyethyl, benzyl, 2-napthylmethyl, O-nitrobenzyl,P-nitrobenzyl, P-methoxybenzyl, 9-phenylxanthyl, trityl (includingmethoxy-trityls), and silyl ethers. Examples of commonly used protectinggroups for sulfhydryls include many of the same protecting groups usedfor hydroxyls. In addition, sulflhydryls can be protected in a reducedform (e.g., as disulfides) or an oxidized form (e.g., as sulfonic acids,sulfonic esters, or sulfonic amides). Protecting groups can be chosensuch that selective conditions (e.g., acidic conditions, basicconditions, catalysis by a nucleophile, catalysis by a lewis acid, orhydrogenation) are required to remove each, exclusive of otherprotecting groups in a molecule. The conditions required for theaddition of protecting groups to amine, alcohol, sulflhydryl, andcarboxylic acid functionalities and the conditions required for theirremoval are provided in detail in T. W. Green and P. G. M. Wuts,Protective Groups in Organic Synthesis (2^(nd) Ed.), John Wiley & Sons,1991 and P. J. Kocienski, Protecting Groups, Georg Thieme Verlag, 1994(hereby incorporated by reference). In the examples that follow, the useof protecting groups is indicated in a structure by the letter P, whereP for any amine, carboxylic acid, sulfhydryl, or alcohol may be any ofthe protecting groups listed above.

EXAMPLE 1 General Coupling Procedure

The synthesis of 5′-substituted benzoxazinorifamycin, 5′-substitutedbenzthiazinorifamycin, and 5′-substituted benzdiazinorifamycin analogscan all proceed through the same general route as shown in Scheme 1,using the general methods disclosed in U.S. Pat. No. 4,965,261 for theattachment of amines to the 5′-position. In this scheme, rifamycinazaquinones of formula II are dissolved in a suitable solvent, forexample, DMSO, and reacted with amines or the formula III in thepresence of manganese dioxide for several hours at room temperature toform azaquinones of formula IV. If required, azaquinones of formula IVcan be further reacted with deprotection reagents to remove X at the25-position, P′ at the 21 and 23 positions, and/or any P″ protectinggroup introduced with amines of formula III. In some embodiments, the25-position can be further derivatized with groups that introduce usefulpharmacodynamic properties, such as groups that transform a rifamycinanalog into a prodrug. Such groups are known to those skilled in theart, examples of which can be found in Testa and Mayer, Hydrolysis inDrug and Prodrug Metabolism: Chemistry, Biochemistry and Enzymology,published by Vch. Verlagsgesellschaft Mbh. (2003), which is herebyincorporated by reference.

A general example is given as follows:

Compound 100 (1.00 g, 1.09 mmol) was dissolved in methyl sulfoxide (10mL) and treated with an amine of formula V (2.18 mmol) and manganese(IV)oxide (0.95 g, 10.9 mmol) for between 12 h to 120 h at rt to 65° C. Thereaction mixture was subsequently diluted with ethyl acetate, filteredthrough celite, washed with water (×3), and dried over Na₂SO₄.Filtration, followed by removal of the solvent in vacuo, yielded aresidue that was purified via flash chromatography (silica gel) using anappropriate solvent system, such as 1-10% methanol in methylenechloride, to produce a benzoxazinorifamycin of formula VI. If furtherpurification is desired, a second column employing C₁₈ silica gel, usingan appropriate solvent system such as 10% water in methanol, wasperformed.

Alternatively, using the methods described in Helv. Chim. Acta 56:2369(1973), a rifamycin quinone of formula VII can be reacted with ananiline of formula VIII, as shown in Scheme 2, to produce an azaquinoneof formula IX.

EXAMPLE 2 General Deacetylation Procedure

A compound of formula VI (˜100 mmol) was dissolved in methanol (5 mL)and then treated with saturated sodium hydroxide in methanol solution (5mL) for between 0.5 h to 3 h at rt. The reaction mixture was then pouredinto saturated ammonium chloride solution and extracted with chloroform.The organics were washed with water (2×) and dried over Na₂SO₄.Filtration, followed by removal of the solvent in vacuo, yielded thedesired des-acetyl product of formula X. If desired, these products werepurified via flash chromatography (silica gel) using an appropriatesolvent system, such as 1-10% methanol in methylene chloride.

EXAMPLE 3 Synthesis of Compound No. 1(3′-hydroxy-5′-(3-hydroxy-1-azetidinyl)benzoxazinorifamycin, See Table 1for Structure)

The title compound is prepared using the general coupling procedure ofExample 1 with compound 100 (1 mmol), 3-hydroxyoxyazetidine, TBDMS ether(2.5 mmol), and manganese(IV) oxide (10 mmol). Removal of the silylprotecting group with tetrabutylammonium fluoride providescompound 1.

EXAMPLE 4 Synthesis of Compound No. 2(25-O-deacetyl-3′-hydroxy-5′-(3-hydroxy-1-azetidinyl)benzoxazinorifamycin)

The title compound is prepared by general deacetylation procedure ofExample 2, using compound 1 to provide compound 2.

EXAMPLE 5 Synthesis of Compound No. 3

The title compound was prepared using the general coupling procedure ofExample 1 with compound 100 (933 mg, 1.02 mmol), commercially available(1S, 4S)-(+)-2,5-diaza-bicyclo[2.2.1]heptane dihydrobromide (531 mg,2.04 mmol), diisopropylethylamine (1.07 mL, 6.12 mmol), andmanganese(IV) oxide (887 mg, 10.2 mmol) to provide compound 3 (206 mg,23% yield) as a blue solid, Mp=>330° C.; ESI(+)MS: 897 (M+H⁺); UV/Vis:λ_(max)=649.4 nm.

EXAMPLE 6 Synthesis of Compound No. 4

The title compound was prepared using the general coupling procedure ofExample 1 using compound 100 (1.27 g, 1.39 mmol),2,5-diaza-bicyclo[2.2.2]octane dihydrochloride (515 mg, 2.78 mmol, J.Heterocyclic Chem. 1974, 449-451 and J. Med. Chem. 1974, 481-487),diisopropylethylamine (1.45 mL, 8.34 mmol), and manganese(IV) oxide(1.21 g, 13.9 mmol) to provide compound 4 (54 mg, 4% yield) as a bluesolid, Mp=>300° C.; ESI(+)MS: 911 (M+H⁺); UV/Vis: λ_(max)=653.2 nm.

EXAMPLE 7 Synthesis of Compound No. 5 (See Table 1 for Structure)

The precursor amine used in the preparation of compound 5 was preparedas follows:

2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate (2.53 g, 8.92 mmol) inabs. ethanol (25 mL) was added to a stirred suspension of compound 101(U.S. Pat. No. 5,654,318, 1.93 g, 8.92 mmol) in aq. Na₂CO₃ (20 mL, 2M),followed by the addition of water (10 mL). The reaction was refluxedwith stirring for 1 hour, cooled, and then stirred under an atmosphereof N₂ at ambient temperature for 19 hours. Most of the solvent wasremoved in vacuo and the resulting slurry was partitioned betweenmethylene chloride and water. The aqueous layer was extracted withmethylene chloride (×2). The organic layers were combined and dried overNa₂SO₄. Filtration followed by removal of the solvent in vacuo yielded aresidue that was purified via flash chromatography (19:1, methylenechloride:methanol) to yield compound 102 (2.59 g, 80% yield) as a lightbrown oil, ¹H NMR (CDCl₃, 300 MHz): 0.03 (s, 9H), 0.95-1.01 (m, 2H),1.38-1.76 (m, 5H), 2.13-2.15 (m, 1H), 2.50 (d, J=8.9 Hz, 1H), 2.61-2.67(m, 1H), 2.73-2.87 (m, 3H), 3.66 (dd, J=21.5, 13.1 Hz, 2H), 3.93 (bs,1H), 4.12-4.17 (m, 2H), 4.62 (bs, 1H), 7.22-7.31 (m, 5H); ESI(+)MS: 361(M+H⁺).

A mixture of compound 102 (2.57 g, 7.12 mmol), Pd/C (1.3 g, 10%, wet),and methanol (50 mL, sparged with H₂) was stirred under balloon pressureH₂ for 18 hours. The reaction was filtered through celite, and thesolvent removed in vacuo. The resulting residue was purified via MPLC(4:1, methylene chloride:methanol) to yield compound 103 (951 mg, 49%yield) as a yellow oil, ¹H NMR (CDCl₃, 300 MHz): 0.04 (s, 9H), 0.98-1.03(m, 2H), 1.36-1.49 (m, 2H), 1.67-1.72 (m, 2H), 1.71 (bs, 1H), 2.00-2.10(m, 1H), 2.73-2.83 (m, 3H), 3.06-3.18 (m, 2H), 4.00 (d, J=12.3 Hz, 1H),4.15-4.21 (m, 2H), 4.49-4.54 (m, 1H); ESI(+) MS: 271 (M+H⁺).

Teoc-protected precursors to compounds 5 and 6 were prepared by generalcoupling procedure 1 using compound 100 (1.56 g, 1.71 mmol), amine 103(922 mg, 3.41 mmol), and manganese(IV) oxide (1.49 g, 17.1 mmol) toprovide 511 mg (28%) of a blue solid (1:1.1, diastereomer A:diastereomer B) and 601 mg (33%) of a blue solid (diastereomer B).Diastereomer A eluted first using a silica column on MPLC (50-100% ethylacetate:hexanes).

A mixture of the Teoc-protected diastereomer A and B precursors tocompound 5 (1:1.1, diastereomer A: diastereomer B, 363 mg, 0.339 mmol),tetrabutylammonium fluoride (3.40 mL, 1.0 M in THF, 3.39 mmol), andacetonitrile (10 mL) was stirred in a sealed flask at 50° C. for 23hours. The reaction was cooled, and poured over water. The aqueous layerwas extracted with chloroform (×2). The combined organics were washedwith water (×3), then dried over Na₂SO₄. Filtration followed by removalof the solvent in vacuo yielded a residue that was purified via MPLC(gradient, 1.25-5% methanol:methylene chloride) followed by preparatorythin layer chromatography (1.0 mm silica, 9:1, methylenechloride:methanol, ˜50 mg per plate) to yield compound 5 as a blue solid(155 mg), Mp=>300° C.; ESI(+)MS: 925 (M+H⁺); UV/Vis: λ_(Xmax)=650.3 nm.

EXAMPLE 8 Synthesis of Compound No. 6 (See Table 1 for Structure)

A mixture of the Teoc-protected diastereomer B precursor to compound 6from Example 10 (337 mg, 0.315 mmol), tetrabutylammonium fluoride (3.15mL, 1.0 M in THF, 3.15 mmol), and acetonitrile (10 mL) was stirred in asealed flask at 50° C. for 23 hours. The reaction was cooled, and pouredover water. The aqueous layer was extracted with chloroform (×2). Thecombined organics were washed with water (×3), then dried over Na₂SO₄.Filtration followed by removal of the solvent in vacuo yielded a residuethat was purified via MPLC (gradient, 1.25 -10% methanol:methylenechloride) followed by preparatory thin layer chromatography (1.0 mmsilica, 9:1, methylene chloride:methanol, ˜50 mg per plate) to yieldcompound 6 as a blue solid (162 mg), Mp=>320° C.; ESI(+)MS: 925 (M+H⁺);UV/Vis: λ_(max)=650.5 nm.

EXAMPLE 9 Synthesis of Compound No. 7 (See Table 1 for Structure)

The title compound was prepared by the general deacetylation procedureof Example 2 using compound 5 (66 mg, 0.0713 mmol) to provide compound 7(62 mg, 98% yield) as a blue solid, Mp=240-243° C.; ESI(+)MS: 883(M+H⁺); UV/Vis: λ_(max)=650.2 nm.

EXAMPLE 10 Synthesis of Compound No. 8 (See Table 1 for Structure)

The title compound was prepared by the general deacetylation procedureof Example 2 using compound 6 (103 mg, 0.111 mmol) to provide 98 mg ofcompound 8 (100% yield) as a blue solid, Mp=216-227° C.; ESI(+)MS: 883(M+H⁺); UV/Vis: λ_(max)=650.3 nm.

EXAMPLE 11 Synthesis of Compound No. 9 (See Table 1 for Structure)

The precursor amine used in the preparation of compound 9 was preparedas follows:

2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate (1.35 g, 4.78 mmol) inacetonitrile (10 mL) was added to a stirred solution of compound 104(Tet. Lett. 2002, 899-902, 920 mg, 4.55 mmol), and diisopropylethylamine(0.833 mL, 4.78 mmol) in acetonitrile (10 mL). The reaction was stirredat ambient temperature for 17.5 hours. The solvent was removed in vacuoand the resulting residue was dissolved in ethyl acetate and washed with1M NaOH (×4), water (×2), then brine (×1), and dried over Na₂SO₄.Filtration, followed by removal of the solvent in vacuo, gave a residuethat was purified via flash chromatography (1:4, ethyl acetate:hexanes,then 1:9 ethyl acetate:hexanes) to produce compound 105 (664 mg, 42%yield) as a colorless oil, ¹H NMR (CDCl₃, 300 MHz): 0.03 (s, 9H),0.96-1.02 (m, 2H), 1.80-1.95 (m, 4H), 2.29 (bs, 2H), 2.59-2.64 (m, 2H),3.46 (s, 2H), 4.15-4.29 (m, 4H), 7.21-7.30 (m, 5H); ESI(+)MS: 347(M+H⁺).

A mixture of compound 105 (645 mg, 1.86 mmol), Pd/C (250 mg, 10%, wet),and methanol (25 mL) was hydrogenated on a Parr apparatus at 50 psi H₂for 5 hours. The reaction was filtered through celite, and the solventremoved in vacuo to yield compound 106 (458 mg, 96% yield) as a whitesolid, ¹H NMR (CDCl₃, 300 MHz): 0.03 (s, 9H), 0.94-0.99 (m, 2H),1.76-1.91 (m, 5H), 2.60-2.64 (m, 2H), 2.94 (bs, 2H), 4.11-4.18 (m, 4H);ESI(+)MS: 257 (M+H⁺), which was used with out further purification.

The Teoc precursor to compound 9 was prepared by the general couplingprocedure of Example 1 using compound 100 (805 mg, 0.880 mmol), amine106 (452 mg, 1.76 mmol), and manganese(IV) oxide (765 mg, 8.80 mmol),which provided a blue solid (875 mg, 94% yield), ESI(+)MS: 1055 (M+H⁺).A mixture of this Teoc precursor (506 mg, 0.480 mmol),tetrabutylammonium fluoride (4.80 mL, 1.0 M in THF, 4.80 mmol), andacetonitrile (15 mL) was stirred in a sealed flask at 50° C. for 22hours. The reaction was cooled, and the solvent removed in vacuo. Theresidue was dissolved in chloroform and washed with water (×3) and driedover Na₂SO₄. Filtration followed by removal of the solvent in vacuoyielded a residue that was purified via MPLC (gradient, 2.5-10%methanol:methylene chloride), followed by reverse phase chromatography(C₁₈ silica, 9:1, methanol:water), followed by preparatory thin layerchromatography (1.0 mm silica, 9:1, methylene chloride:methanol, ˜50 mgper plate) to yield compound 9 as a blue solid (198 mg, 45% yield),Mp=210-212° C.; ESI(+)MS: 911 (M+H⁺); UV/Vis: λ_(max)=640.9 nm.

EXAMPLE 12 Synthesis of Compound No. 10 (See Table 1 for Structure)

The title compound was prepared by the general deacetylation procedureof Example 2 using compound 9 (87 mg, 0.0955 mmol) to provide compound10 (83 mg, 100% yield) of a blue solid, Mp=208-229° C.; ESI(+)MS: 869(M+H⁺); UV/Vis: λ_(max)=641.2 nm.

EXAMPLE 13 Synthesis of Compound No. 11 (See Table 1 for Structure)

The precursor amine used in the preparation of compound 11 was preparedas follows:

Ethyl chloroformate (0.491 mL, 5.14 mmol) was added to a stirredsolution of compound 101 (1.01 g, 4.67 mmol, U.S. Pat. No. 5,654,318),triethylamine (0.980 mL, 7.01 mmol), and methylene chloride (20 mL) at0° C. The reaction was stirred under an atmosphere of N₂ at 0° C. for 30minutes, quenched with sat. NaHCO₃ and the aqueous layer extracted withmethylene chloride (×1). The organics were combined and dried overNa₂SO₄. Filtration, followed by removal of the solvent in vacuo, yieldeda residue that was purified via flash chromatography (1:1, ethyl acetatehexanes) to yield compound 107 (1.06 g, 79% yield) as a colorless oil,¹H NMR (CDCl₃, 300 MHz): 1.24 (t, J=7.1 Hz, 3H), 1.37-1.72 (m, 4H),2.10-2.16 (m, 1H), 2.51 (d, J=8.9 Hz, 1H), 2.64 (t, J=8.2 Hz, 1H),2.75-2.84 (m, 3H), 3.66 (dd, J=24.2, 13.1 Hz, 2H), 3.92 (bs, 1H), 4.11(q, J=7.1 Hz, 2H), 4.63 (bs, 1H), 7.22-7.32 (m, 5H); ESI(+)MS: 289 (M+H+).

A mixture of compound 107 (1.05 g, 3.64 mmol), Pd/C (300 mg, 10%, wet),and methanol (25 mL) was hydrogenated on a Parr apparatus at 50 psi H₂for 14.5 hours. The reaction was filtered through celite, and thesolvent removed in vacuo to yield compound 108 (767 mg, quant., wet withmethanol) as a light yellow oil, 1H NMR (CDCl₃, 500 MHz): 1.27 (t, J=3.5Hz, 3H), 1.38-1.45 (m, 2H), 1.68-1.73 (m, 2H), 2.04-2.07 (m, 2H),2.75-2.83 (m, 3H), 3.09-3.18 (m, 2H), 4.00 (d, J=11.5 Hz, 1H), 4.11-4.17(m, 2H), 4.52-4.54 (m, 1H); ESI(+)MS: 199 (M+H⁺), which was used without further purification.

The title compound was prepared by the general coupling procedure ofExample 1 using compound 100 (1.65 g, 1.80 mmol), amine 108 (712 mg,3.59 mmol) and manganese(IV) oxide (1.56 g, 18.0 mmol) to providecompound 11, Diastereomer A (555 mg (31%) as a blue solid (higher R_(f)using ethyl acetate as eluent) and compound 12, Diastereomer B (660 mg,37% yield, not shown in Table) as a blue solid (lower R_(f) using ethylacetate as eluent), Mp=>300° C.; ESI (+)MS: 997 (M+H⁺); UV/Vis:λ_(max)=644.0 nm.

EXAMPLE 14 Synthesis of Compound No. 13 (See Table 1 for Structure)

The title compound was prepared by the general deacetylation procedureof Example 2 using compound 11 (110 mg, 0.110 mmol) to provide compound13 (97 mg, 92% yield) as a blue solid, as mixture of diastereomers (asdetermined by HPLC and ¹H NMR), Mp=>300° C.; ESI(+)MS: 955 (M+H⁺);UV/Vis: λ_(max)=644.0 nm.

EXAMPLE 15 Synthesis of Compound No. 14 (See Table 1 for Structure)

The precursor amine used in the preparation of compound 47 was preparedas follows:

Acetyl chloride (0.373 mL, 5.24 mmol) was added to a stirred solution ofcompound 101 (1.03 g, 4.76 mmol), triethylamine (1.0 mL, 7.14 mmol), andmethylene chloride (20 mL) at 0° C. The reaction was stirred under anatmosphere of N₂ at 0° C. for 1 hour. The reaction was quenched withsat. NaHCO₃, and the aqueous layer was extracted with methylene chloride(×1). The organics were combined and dried over Na₂SO₄. Filtrationfollowed by removal of the solvent in vacuo yielded a residue that waspurified via flash chromatography (9:1, methylene chloride:methanol) toyield compound 109 (1.09 g, 87%) as a light yellow oil, ¹H NMR (CDCl₃,300 MHz): 1.31-1.78 (m, 4H), 2.06-2.39 (m, 4H), 2.48-2.90 (m, 4.5H,conformer), 3.06-3.16 (m, 0.5H, conformer), 3.58-3.75 (m, 2.5H,conformer), 4.32-4.44 (m, 1H), 5.03 (q, J=8.5 Hz, 0.5H, conformer),7.21-7.35 (m, 5H); ESI(+)MS: 259 (M+H⁺).

A mixture of compound 109 (2.18 g, 8.43 mmol), Pd/C (500 mg, 10%, wet),and methanol (50 mL) was hydrogenated on a Parr apparatus at 50 psi H₂for 15 hours. The reaction was filtered through celite, and the solventremoved in vacuo to yield compound 110 (1.55 g, quant., wet withmethanol) as a light yellow oil, ¹H NMR (CDCl₃, 500 MHz): 1.35-1.53 (m,2H), 1.73-1.77 (m, 2H), 2.08-2.14 (m, 4H), 2.56 (t, J=10.0 Hz, 0.4H,conformer), 2.77 (t, J=10.4 Hz, 0.6H, conformer), 2.82-2.86 (m, 2H),2.96 (t, J=10.0 Hz, 0.4H, conformer), 2.82-2.86 (m, 2H), 3.05-3.09 (m,1H), 3.16-3.25 (m, 1.6H, conformer), 3.66 (d, J=12.7 Hz, 0.6H,conformer), 4.27 (q, J=8.1 Hz, 0.4H, conformer), 4.51 (d, J =13.9 Hz,0.4H, conformer), 4.97 (q, J=8.1 Hz, 0.6H, conformer); ESI(+)MS: 169(M+H⁺), that was used with out further purification.

The title compound was prepared by the general coupling procedure ofExample 1 using compound 100 (3.81 g, 4.16 mmol), amine 110 (1.40 g,8.32 mmol) and manganese(IV) oxide (3.62 g, 41.6 mmol) to providecompound 14 (916 mg, 23% yield) as a blue solid, Mp=148-149° C.;ESI(+)MS: 967 (M+H⁺); UV/Vis: λ_(max)=644.0 nm.

EXAMPLE 16 MIC Assay

MICs of candidate compounds of the invention can be determined, forexample, by the method of Lee et al., Am. Rev. Respir. Dis. 136:349(1987). To a BACTEC 12B vial (4 mL of 7H 12B medium), 0.1 mL of a10-fold dilution of subculture of the test organisms in 7H9 medium(optical density at 540 nm, 0.1) is inoculated and cultured at 37° C.until a growth index (GI) of 999 is reached. Then the broth culture isremoved and diluted 100-fold, and 0.1 mL of the dilution is inoculatedinto a BACTEC 12B vial with or without a candidate compound. Thecandidate compound containing vials can hold 0.1 mL of candidatecompound solution appropriately diluted to obtain the desiredconcentration. A 1% control vial, 0.1 mL of the 100-fold dilution of theinoculum described above, is inoculated into 12B vial without candidatecompound. The 12B vials are incubated at 37° C., and GI readingsrecorded daily, using a BACTEC 460 TB instrument (Johnston Laboratories,Townsend, Md.), until the control vial reaches a GI greater than 30.When the final readings in the GI of the candidate containing vials arelower than those of the 1% control, the drug is considered to haveinhibited more than 99% of the bacterial population, and thisconcentration will be defined as the MIC.

Table 1 gives MIC values for some of the compounds of the invention.TABLE 1 Structures and MIC values MIC (μg/mL) Compound S. S. E. No.Structure* MW MP (° C.) aureus pneumo. faecalis H. flu E. coli 1

875.99 0.002 0.00012 0.12 0.12 4 5

925.039 >300 0.015 0.00025 0.5 2 8 7

883.002 240-243 0.12 0.004 4 4 >8 6

925.039 >320 0.004 0.00025 0.12 0.25 8 8

883.002 216-227 0.03 0.002 0.25 1 >8 9

911.012 210-212 0.008 0.00012 1 2 >8 10

868.975 208-229 0.015 0.001 0.12 0.25 4 11

997.102 >300 0.06 0.008 1 0.5 >8 13

955.065 >300 0.008 0.00012 2 0.5 8 14

967.076 148-149 0.008 0.002 1 0.25 >8 15

925.039 148-150 0.004 0.00006 2 0.5 >8 *A′ and B′ represent thefollowing moieties: A′ is

and B′ is

OTHER EMBODIMENTS

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

While the invention has been described in connection with specificembodiments, it will be understood that it is capable of furthermodifications. Therefore, this application is intended to cover anyvariations, uses, or adaptations of the invention that follow, ingeneral, the principles of the invention, including departures from thepresent disclosure that come within known or customary practice withinthe art.

Other embodiments are within the claims.

1. a compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein A is H, OH,O—(C₁₋₆ alkyl), O—(C₁₋₄ alkaryl), O—(C₆₋₁₂ aryl), O—(C₁₋₉ heteroaryl),or O—(C₁₋₄ alkheteroaryl); W is O, S, or NR¹, wherein R¹ is H, C₁₋₆alkyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl; X is H or COR², wherein R²is C₁₋₆ alkyl, which can be substituted with 1-5 OH groups, O—(C₃₋₇alkyl), which can be substituted with 1-4 OH groups, C₆₋₁₂ aryl, C₁₋₄alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl, wherein each alkylcarbon is bonded to to no more than one oxygen atom; Y is H, Hal, orOR^(Y3), wherein R^(Y3) is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉heteroaryl, or C₁₋₄ alkheteroaryl; Z is H, Hal, or OR^(Z3), whereinR^(Z3) is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄alkheteroaryl; and R⁴ has the formula:

 wherein R⁵ is H, C₁₋₆ alkyl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, COR¹⁰,CO₂R¹¹, CONR¹⁰ R¹¹ CSR¹⁰, COSR¹¹, CSOR¹¹, CSNR¹⁰ R¹¹, SO₂R¹¹, orSO₂NR¹⁰R¹¹, wherein R¹⁰ is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉heteroaryl, or C₁₋₄ alkheteroaryl, R¹¹ is C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₄alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl, or R¹⁰ and R¹¹ togetherform a C₂₋₆ linkage, optionally containing a non-vicinal O; R⁶ is H,C₁₋₆ alkyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl; R⁷ is H, C₁₋₆ alkyl,C₆₋₁₂ aryl, C₁₋₉ heteroaryl, C₂₋₉ heterocyclyl, C₁₋₄ alkaryl, C₁₋₄alkheteroaryl, OR¹², or NR¹²R¹³, wherein R¹² is H, C₁₋₆ alkyl, C₆₋₁₂aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl, R¹³ is C₁₋₆alkyl, C₆₋₁₂ aryl, C₁₋₄ alkaryl, C₁₋₉ heteroaryl, or C₁₋₄ alkheteroaryl,or R¹² and R¹³ together forn a C₂₋₆ linkage, optionally containing anon-vicinal O; T is O, S, NR⁵, or a bond; each of R⁸ and R⁹ is,independently H, C₁₋₆ alkyl, C₆₋₁₂ aryl, C₁₋₉ heteroaryl, C₂₋₉heterocyclyl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, or R⁸ and R⁵ togetherform a 3-8-membered ring, with the ring optionally containing anon-vicinal oxygen; and each of r and s is, independently, 1 or
 2. 2.The compound of claim 1, wherein said compound is selected from thegroup consisting of:


3. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent and the compound of claim
 1. 4. A method of treatingor preventing a microbial infection in an animal, or a condition causedby a microbial infection in an animal, said method comprisingadministering to said animal the compound of claim 1 in an amountsufficient to treat or prevent said microbial infection.
 5. The methodof claim 4, wherein said said compound is administered orally,topically, intravenously, intramuscularly, or subcutaneously.
 6. Themethod of claim 4, wherein said animal is a human.
 7. The method ofclaim 4, wherein said condition is selected from the group consisting ofcommunity-acquired pneumonia, upper and lower respiratory tractinfections, skin and soft tissue infections, bone and joint infections,and hospital-acquired lung infections.
 8. The method of claim 4, whereinsaid infection is a persistent infection.
 9. The method of claim 4,wherein said infection is an ear infection.
 10. The method of claim 4,wherein said microbial infection is a protozoan, bacterial, viral, orfungal infection.
 11. The method of claim 4, wherein said microbialinfection is an intracellular infection.
 12. The method of claim 4,wherein said infection is caused by a Gram-positive coccus.
 13. Themethod of claim 4, wherein said infection is caused by N. gonorrhoeae.14. The method of claim 4, wherein said infection is caused by C.difficile.
 15. The method of claim 4, wherein said infection is causedby C. pneumoniae.
 16. The method of claim 4, wherein said infection iscaused by H. pylori.
 17. The method of claim 16, wherein said methodfurther comprises administering to said animal a proton pump inhibitoror bismuth preparation.
 18. The method of claim 4, wherein saidmicrobial infection is caused by a bacterium selected from the groupconsisting of Anaplasma bovis, A. caudatum, A. centrale, A. marginale A.ovis, A. phagocytophila, A. platys, Bartonella bacilliformis, B.clarridgeiae, B. elizabethae, B. henselae, B. henselae phage, B.quintana, B. taylorii, B. vinsonii, Borrelia afzelii, B. andersonii, B.anserina, B. bissettii, B. burgdorferi, B. crocidurae, B. garinii, B.hermsii, B. japonica, B. miyamotoi, B. parkeri, B. recurrentis, B.turdi, B. turicatae, B. valaisiana, Brucella abortus, B. melitensis, C.psittaci, C. trachomatis, Cowdria ruminantium, Coxiella burnetii,Ehrlichia canis, E. chaffeensis, E. equi, E. ewingii, E. muris, E.phagocytophila, E. platys, E. risticii, E. ruminantium, E. sennetsu,Haemobartonella canis, H. felis, H. muris, Mycoplasma arthriditis, M.buccale, M. faucium, M. fermentans, M. genitalium, M. hominis, M.laidlawii, M. lipophilum, M. orale, M. penetrans, M. pirum, Mpneumoniae, M. salivarium, M. spermatophilum, Rickettsia australis, R.conorii, R. felis, R. helvetica, R. japonica, R. massiliae, R.montanensis, R. peacockii, R. prowazekii, R. rhipicephali, R.rickettsii, R. sibirica, and R. typhi.
 19. The method of claim 4,wherein said method further comprises administering to said animal asecond agent selected from the group consisting of: antifungal agent,antiviral agent, antibacterial agent, and antiprotozoan agent.
 20. Amethod of treating or preventing the development of anatherosclerosis-associated disease in a patient in a patient, saidmethod comprising administering to said patient the compound of claim 1in an amount sufficient to treat or prevent saidatherosclerosis-associated disease.
 21. The method of claim 20, whereinsaid method further comprises administering to said animal a secondagent selected from the group consisting of anti-inflammatory agent,antibacterial agent, platelet aggregation inhibitor, anticoagulant,antipyretic, or lipid lowering agent.
 22. The method of claim 20,wherein said patient is diagnosed as having saidatherosclerosis-associated disease.